A method for preparing a coral-like SERS active substrate, a coral-like SERS active substrate and its application

Abstract

The present invention relates to the technical field of surface enhanced Raman scattering substrates, and discloses a method for preparing a coral-like SERS active substrate, a coral-like SERS active substrate, and applications thereof. The coral-like SERS active substrate prepared by the method provided by the present invention has a very high SERS enhancement activity, can be used for rapid detection of edible pigments, and the active substrate also has the advantage of low cost; at the same time, the coral-like SERS active substrate provided by the present invention is easy to operate and more universal. The method for preparing a coral-like SERS active substrate provided by the present invention can simplify the nanoparticle preparation process; and can realize in-situ preparation at the detection site without the need for storage and transportation; at the same time, the preparation method also has the advantages of high reproducibility, easy operation, and low cost, and has good application prospects in on-site rapid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of surface enhanced Raman scattering substrates, and in particular to a method for preparing a coral-shaped SERS active substrate, the coral-shaped SERS active substrate and applications thereof. Background Art

[0002] In recent years, as consumers pay more attention to food safety and healthy eating, the safety of food coloring has been controversial. Although the dosage of food coloring added to food is very small, people consume a variety of foods containing different pigments every day. Long-term and cumulative intake of these pigments will accumulate in the human body, causing potential risks.

[0003] Studies have shown that food coloring is a major cause of allergic reactions, ADHD, and reproductive system problems in children. It can also damage hippocampal nerve growth, inhibit enzyme activity in red blood cells, and cause diarrhea and poisoning. However, these pigments remain popular and widely used due to their bright colors, stable properties, and low price. The problem of food coloring overuse has remained prominent in recent years.

[0004] Therefore, it is very important to establish a convenient, rapid, efficient, sensitive and accurate method for detecting food pigments. Surface enhanced Raman scattering (SERS) is a detection method that has the above advantages.

[0005] SERS analysis has become a promising trace detection method due to its low cost, high sensitivity, simplicity, and ability to be performed on-site, rapidly, and non-destructively. However, current SERS analysis typically requires pre-preparation of an enhanced substrate, a process that is generally subject to the following challenges:

[0006] The nanoparticle preparation process is very cumbersome and time-consuming, and there are large differences in the uniformity and stability of nanoparticles prepared from different batches; nanoparticles are very prone to aggregation after multiple treatments, and their structure and dispersion are also prone to changes during storage; the preparation of the reinforced substrate is prone to produce the coffee ring effect, which makes it difficult to ensure its uniformity.

[0007] The above problems will greatly affect the reproducibility and reliability of the SERS analysis method. Therefore, it is of great practical value to develop a SERS substrate that can be quickly prepared on-site for rapid detection of targets. Summary of the Invention

[0008] The purpose of the present invention is to overcome the defects of the existing method for preparing SERS substrates, such as the complicated preparation process, long time consumption and poor uniformity and stability of the prepared SERS substrates.

[0009] In order to achieve the above object, a first aspect of the present invention provides a method for preparing a coral-like SERS active substrate, the method comprising:

[0010] In the presence of a protective agent and solvent I, silver nitrate and a copper sheet are subjected to an in-situ reduction reaction to obtain the coral-like SERS active substrate;

[0011] The protective agent is fish sperm DNA (FSDNA); the solvent I is water.

[0012] The second aspect of the present invention provides a coral-like SERS active substrate prepared by the method described in the first aspect.

[0013] The third aspect of the present invention provides a use of the coral-shaped SERS active substrate described in the second aspect in detecting food pigments.

[0014] A fourth aspect of the present invention provides a method for measuring the content of food pigments, the method comprising:

[0015] (1) In the presence of solvent II, cysteamine hydrochloride (CA) and food coloring are mixed to obtain a mixture I;

[0016] (2) contacting the mixture I with a coral-like SERS active substrate;

[0017] (3) establishing a standard curve using SERS spectroscopy to determine the content of the food pigment;

[0018] Wherein, the food coloring is selected from at least one of allura red, brilliant blue, and sunset yellow;

[0019] The coral-like SERS active substrate is the coral-like SERS active substrate described in the second aspect.

[0020] Compared with the prior art, the present invention has at least the following advantages:

[0021] (1) The silver nanoparticles in the coral-like SERS active substrate provided by the present invention are evenly distributed on the surface of the copper sheet in a coral-like shape. The coral branches have a rough, uneven structure with small spacing between the branches, which can produce strong plasmon coupling and provide a large number of SERS "hot spots", thereby having high SERS enhancement activity and being able to be used for rapid detection of food coloring. In addition, the active substrate has the advantage of low cost. At the same time, the coral-like SERS active substrate provided by the present invention is easy to operate and more universal.

[0022] (2) The method for preparing a coral-like SERS active substrate provided by the present invention can simplify the nanoparticle preparation process, thereby greatly reducing the time it takes for the prepared silver nanoparticles to be oxidized; and can achieve in-situ preparation at the detection site without the need for storage and transportation, thereby overcoming the difficulty of the silver nanoparticle substrate being easily oxidized; at the same time, the preparation method also has the advantages of high reproducibility, easy operation, and low cost, and has good application prospects in on-site rapid detection;

[0023] (3) The method for preparing coral-like SERS active substrate provided by the present invention utilizes Ag + Replacement reaction with Cu, the generated Cu 2+ The in situ generation of silver nanoparticles on the network formed by cross-linking with FSDNA can avoid the coffee ring effect and make the prepared coral-like SERS activity of the substrate more uniform and reproducible.

[0024] (4) The method for determining the content of food pigments provided by the present invention utilizes the property of CA to form hydrogen bonds with sulfonic acid groups, and only captures pigments with sulfonic acid groups (weak affinity molecules) for SERS analysis, thereby greatly improving the sensitivity and selectivity of detection, and providing a new idea for the qualitative and quantitative determination of such weak affinity molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of a preferred SERS active substrate preparation and allura red pigment detection process provided by the present invention;

[0026] Figure 2 is a data graph provided by the present invention to prove the ability of copper sheet to enhance the SERS active substrate; wherein, Figure 2 a is a photo of the silver nanoparticles being peeled off from the copper sheet and pasted onto the glass sheet. Figure 2 b is the Raman spectrum change diagram of different SERS active substrates. Figure 2 c is the SERS activity of different substrates at 634 cm -1 SERS intensity error bar diagram at ;

[0027] Figure 3 The following are the physical pictures, SEM and mapping pictures of SERS active substrates prepared with different types of protective agents provided by the present invention; Figure 3 a is the physical image, SEM and mapping image of the SERS active substrate prepared by FSDNA. Figure 3 b is the physical image, SEM and mapping image of the SERS active substrate prepared by hexadecyltrimethylammonium chloride. Figure 3c is the physical image, SEM and mapping image of the SERS active substrate prepared with polyvinyl pyrrolidone. Figure 3 d is the physical image, SEM and mapping image of the SERS active substrate prepared with bovine serum albumin;

[0028] Figure 4 The Raman spectra of the SERS active substrate prepared by different protective agent dosages, different silver nitrate dosages and different in-situ reduction reaction times provided by the present invention and the Raman spectra at 634 cm -1 SERS intensity error bar diagram at ; where, Figure 4 a1 and Figure 4 a2 are the Raman spectra of SERS active substrates prepared with different amounts of protective agents and the Raman spectra at 634 cm -1 SERS intensity error bar diagram at Figure 4 b1 and Figure 4 b2 are the Raman spectra of SERS active substrates prepared with different amounts of silver nitrate and the Raman spectra at 634 cm -1 SERS intensity error bar diagram at Figure 4 c1 and Figure 4 c2 are the Raman spectra of the SERS active substrates prepared with different in situ reduction reaction times and the Raman spectra at 634 cm -1 SERS intensity error bar diagram at ;

[0029] Figure 5 The present invention provides a preferred Raman spectrum of allura red pigment detected by using different cysteamine hydrochloride dosages, different first reaction times, different pH values ​​and different contact reaction times, as well as the Raman spectrum at 1267 cm -1 SERS intensity error bar diagram at ; where, Figure 5 a1 and Figure 5 a2 are the Raman spectra of allura red pigment detected by different first reaction times and the Raman spectra at 1267 cm -1 Error bars of SERS intensity at Figure 5 b1 and Figure 5 b2 are the Raman spectra of allura red pigment detected by different cysteamine hydrochloride dosages and the Raman spectra at 1267 cm -1 SERS intensity error bar diagram at Figure 5 c1 and Figure 5 c2 are the Raman spectra of allura red pigment detected by different contact reaction times and the Raman spectra at 1267 cm -1 SERS intensity error bar diagram at Figure 5 d1 and Figure 5 d2 are the Raman spectra of allura red pigment detected at different pH values ​​and the Raman spectra at 1267 cm-1 SERS intensity error bar diagram at ;

[0030] Figure 6 is a data graph provided by the present invention to prove the uniformity of the SERS active substrate; wherein, Figure 6 a is the 634 cm-1 peak at three random points on ten different batches of SERS active substrates. -1 SERS intensity error bar diagram, Figure 6 b is the 634 cm-1 peak at 20 random points on the first batch of SERS active substrates. -1 SERS intensity error bar diagram;

[0031] Figure 7 is a Raman spectrum diagram for calculating the SERS active substrate enhancement factor provided by the present invention; wherein, Figure 7 a is the Raman spectrum of the SERS active substrate G1 containing 0.1 mol / L 4-MBPA aqueous solution, Figure 7 b is the Raman spectrum of the SERS active substrate G1 containing 0.01 μmol / L 4-MBPA aqueous solution;

[0032] Figure 8 It is the SERS intensity error bar graph of different food pigments detected by the method provided by the present invention;

[0033] Figure 9 The Raman spectra and standard linear curves of the method provided by the present invention for detecting different contents of edible pigments are shown; wherein, Figure 9 a1 and Figure 9 a2 are the Raman spectra and standard linear curves for detecting different contents of brilliant blue pigment, Figure 9 b1 and Figure 9 b2 are the Raman spectra and standard linear curves for detecting different contents of allura red pigment, Figure 9 c1 and Figure 9 Raman spectra and standard linear curves of sunset yellow pigments with different contents detected by c2;

[0034] Figure 10 The Raman spectra of food pigments in different actual samples detected by the method provided by the present invention are as follows;

[0035] Figure 11 This is a Raman spectrum of the spiked recovery of food pigments in artificial urine provided by the present invention; wherein, Figure 11 a is the Raman spectrum of the spiked recovery of brilliant blue pigment in artificial urine. Figure 11 b is the Raman spectrum of sunset yellow pigment spiked and recovered in artificial urine. Figure 11 c is the Raman spectrum of spiked recovery of allura red pigment in artificial urine;

[0036] Figure 12 : is a length and diameter distribution diagram of silver nanoparticles on a SERS active substrate prepared with different protective agents provided by the present invention; wherein, Figure 12 a1 is the diameter distribution of silver nanoparticles on the SERS active substrate prepared by bovine serum albumin, Figure 12 a2 is the length distribution of silver nanoparticles on the SERS active substrate prepared with bovine serum albumin; Figure 12 b1 is the diameter distribution diagram of silver nanoparticles on the SERS active substrate prepared by hexadecyltrimethylammonium chloride, Figure 12 b2 is the length distribution of silver nanoparticles on the SERS active substrate prepared by hexadecyltrimethylammonium chloride; Figure 12 c1 is the diameter distribution of silver nanoparticles on the SERS active substrate prepared by FSDNA, Figure 12 c2 is the length distribution of silver nanoparticles on the SERS active substrate prepared by FSDNA; Figure 12 d1 is the diameter distribution of silver nanoparticles on the SERS active substrate prepared by polyvinyl pyrrolidone, Figure 12 d2 is the length distribution of silver nanoparticles on the SERS active substrate prepared by polyvinyl pyrrolidone. DETAILED DESCRIPTION

[0037] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0038] As mentioned above, the first aspect of the present invention provides a method for preparing a coral-like SERS active substrate, the method comprising:

[0039] In the presence of a protective agent and solvent I, silver nitrate and a copper sheet are subjected to an in-situ reduction reaction to obtain the coral-like SERS active substrate;

[0040] The protective agent is fish sperm DNA; the solvent I is water.

[0041] In the present invention, the silver nanoparticles in the SERS-active substrate are evenly distributed on the copper surface in a coral-like pattern. The coral branches have a rough, uneven structure with small spacing between branches, which enables strong plasmon coupling and provides a large number of SERS "hotspots," resulting in excellent SERS enhancement. The inventors of the present invention also found that when the silver nanoparticles are peeled from the copper sheet and pasted onto a glass sheet, their SERS enhancement ability is greatly weakened; however, when the silver nanoparticles are then pasted back onto the copper sheet, their SERS enhancement ability is restored. See the specific results for details. Figure 2 .

[0042] Figure 2 This is a data graph provided by the present invention to prove the copper sheet's ability to enhance the SERS active substrate. Figure 2 It can be seen from the figure that there is also plasmon coupling between the silver nanoparticles and the copper sheet.

[0043] In the present invention, the inventors have studied different types of protective agents, and the specific results are shown in Figure 3 .

[0044] Figure 3 The following are the physical pictures, SEM and mapping pictures of SERS active substrates prepared with different types of protective agents provided by the present invention. Figure 3 It can be seen that the SERS active substrate prepared using FSDNA as a protective agent is more uniform than the SERS active substrate prepared using cetyltrimethylammonium chloride (CTAC), polyvinylpyrrolidone (PVP) and bovine serum albumin (BSA).

[0045] Since FSDNA is a short-chain DNA, it has a large number of 2+ The coordinated groups can form a cross-linked network structure, which can be used as a soft membrane to adsorb the generated silver nanoparticles and make the silver nanoparticles distributed in a coral-like manner, thereby showing the most uniform SERS enhancement ability. Although BSA also contains some groups similar to FSDNA, its size is much larger than FSDNA. Due to the influence of steric hindrance, it is difficult to form a high-density network, so the prepared SERS active substrate is uneven. Although PVP and CTAC are both commonly used ligands to protect nanoparticles, neither of them has the same SERS enhancement ability as Cu. 2+ The silver nanoparticles are difficult to be adsorbed and are not strong enough to fall off, which results in poor uniformity of the prepared SERS active substrate.

[0046] Preferably, relative to 100 μL of the solvent I, the amount of the protective agent used is 12-75 μg, and the amount of the silver nitrate used is 3-6 μmol.

[0047] More preferably, the amount of the protective agent is 50-75 μg, and the amount of the silver nitrate is 4-5 μmol relative to 100 μL of the solvent I. In this preferred embodiment, the prepared coral-like SERS active substrate has better uniformity and more excellent SERS enhancement activity.

[0048] Preferably, the in-situ reduction reaction conditions at least meet the following requirements: temperature of 20-30° C., and time of 3-20 min.

[0049] More preferably, the in-situ reduction reaction conditions at least meet the following requirements: temperature of 20-30° C. and time of 3-10 min. In this preferred embodiment, the prepared coral-like SERS active substrate has better uniformity and more excellent SERS enhancement activity.

[0050] In the present invention, the inventors studied the amount of protective agent, the amount of silver nitrate and the in-situ reduction reaction time, and the specific results are shown in Figure 4 .

[0051] Figure 4 The Raman spectra of the SERS active substrate prepared by different protective agent dosages, different silver nitrate dosages and different in-situ reduction reaction times provided by the present invention and the Raman spectra at 634 cm -1 SERS intensity error bar diagram at . Figure 4 It can be seen that the amount of protective agent and silver nitrate as well as the in situ reduction reaction time have a great influence on the activity of the prepared SERS active substrate.

[0052] Too much protective agent and too little silver nitrate will cause the protective agent to + Cu replaced by Cu 2+ Excessive cross-linking occurs, causing the replaced silver nanoparticles to be almost completely covered by the network formed by cross-linking, resulting in a weakened SERS enhancement ability of the silver nanoparticles; while too little protective agent and too much silver nitrate will make the cross-linked network insufficient to adsorb all the silver nanoparticles, causing the silver nanoparticles to fall off and making the prepared SERS active substrate uneven; the distribution shape of silver nanoparticles will change with the increase of in situ reduction reaction time, and the SERS enhancement activity and uniformity of the SERS active substrate are directly related to the distribution shape of silver nanoparticles.

[0053] The preparation method of the present invention may also include various post-processing methods known in the art, such as washing and drying. The present invention does not particularly limit the post-processing steps. For example, the present invention may first wash and dry the coral-shaped SERS-active substrate obtained after the in-situ reduction reaction. Exemplarily, the drying conditions may be a drying temperature of 30-50°C and a drying time of 0.1-2 minutes.

[0054] As mentioned above, the second aspect of the present invention provides a coral-like SERS active substrate prepared by the method described in the first aspect.

[0055] As mentioned above, the third aspect of the present invention provides a use of the coral-shaped SERS active substrate described in the second aspect in detecting food pigments.

[0056] As mentioned above, the fourth aspect of the present invention provides a method for determining the content of food pigments, the method comprising:

[0057] (1) in the presence of solvent II, reacting cysteamine hydrochloride with food coloring to obtain a mixture I;

[0058] (2) contacting the mixture I with a coral-like SERS active substrate;

[0059] (3) establishing a standard curve using SERS spectroscopy to determine the content of the food pigment;

[0060] Wherein, the food coloring is selected from at least one of allura red, brilliant blue, and sunset yellow;

[0061] The coral-like SERS active substrate is the coral-like SERS active substrate described in the second aspect.

[0062] It should be noted that the present invention has no particular requirements for the type of solvent II. For example, the solvent II can be ultrapure water. Ultrapure water refers to water from which the conductive medium is almost completely removed and from which undissociated colloidal substances, gases, and organic matter are removed to very low levels.

[0063] In the present invention, the inventors studied the dosage of cysteamine hydrochloride, the first reaction time, the pH value and the contact reaction time. The specific results are shown in Figure 5 .

[0064] Figure 5 The present invention provides a preferred Raman spectrum of allura red pigment detected by using different cysteamine hydrochloride dosages, different first reaction times, different pH values ​​and different contact reaction times, as well as the Raman spectrum at 1267 cm-1 SERS intensity error bar diagram at . Figure 5 It can be seen that the amount of cysteamine hydrochloride, the first reaction time, the pH value and the contact reaction time all have a great influence on the accuracy of quantitative detection of food pigments.

[0065] Appropriate CA dosage and appropriate first reaction time can enable more food coloring to be captured by CA; and appropriate contact reaction time can enable more "CA-food coloring" to form Ag-S bonds with the SERS active substrate and thus be fixed on the substrate; when the pH value is too low, the free hydrogen ions increase, which will compete with the hydrogen on the amino group of CA, thereby affecting the formation of hydrogen bonds; when the pH value is too high, the increase in free hydroxide will affect the almost exposed hydrogen in the amino group of CA, thereby affecting the formation of hydrogen bonds.

[0066] According to a preferred embodiment, relative to 10 μL of the solvent II, the amount of the cysteamine hydrochloride used is 0.05-0.20 μmol, and the amount of the food coloring used is 0.001-0.1 μg.

[0067] More preferably, the amount of cysteamine hydrochloride used is 0.10-0.20 μmol, and the amount of food pigment used is 0.01-0.1 μg relative to 10 μL of solvent II. In this preferred embodiment, the food pigment content result obtained by this method is more accurate.

[0068] Preferably, the conditions of the first reaction at least meet the following requirements: the reaction is carried out under stirring, with a stirring speed of 4000-5000 rpm, a stirring time of 0.5-10 min, and a pH value of 2.30-6.20.

[0069] Further preferably, the conditions of the first reaction at least meet the following requirements: the reaction is carried out under stirring, with a stirring speed of 4000-5000 rpm, a stirring time of 1-10 min, and a pH value of 2.30-5.30. In this preferred embodiment, the content of the food pigment detected by this method is more accurate.

[0070] Preferably, the contact reaction conditions at least meet the following requirements: temperature of 20-30° C. and time of 1-30 min.

[0071] More preferably, the contact reaction conditions at least meet the following requirements: temperature of 20-30° C. and time of 5-20 min. In this preferred embodiment, the food pigment content detected by this method is more accurate.

[0072] The present invention will be described in detail below through examples.

[0073] In the following examples, unless otherwise specified, all raw materials used are commercially available products.

[0074] Unless otherwise specified, room temperature in the present invention means 25±2°C.

[0075] Cysteamine hydrochloride: brand C804763, purchased from Maclean Company.

[0076] FSDNA: Brand D3159, purchased from Sigma.

[0077] Silver nitrate: analytical grade, brand 10018464, purchased from Sinopharm.

[0078] CTAC: Brand A610332, purchased from Sangon Biotech Co., Ltd.

[0079] PVP: brand 80154482, purchased from Sinopharm Group.

[0080] BSA: Brand A116563, purchased from Aladdin.

[0081] 4-MBPA: 90% pure, purchased from Sigma.

[0082] Allura red pigment: brand E107191, purchased from Aladdin.

[0083] Brilliant blue pigment: brand E101979, purchased from Aladdin.

[0084] Sunset yellow pigment: brand S109563, purchased from Aladdin Company.

[0085] Erythrosin: brand E107191, purchased from Aladdin.

[0086] New red pigment: brand N886279, purchased from Maclean Company.

[0087] Amaranth pigment: brand A800746, purchased from Maclean Company.

[0088] Indigo pigment: brand I104182, purchased from Aladdin Company.

[0089] Anthocyanin pigment: brand C885847, purchased from Maclean Company.

[0090] Curcumin: brand C805205, purchased from Maclean Company.

[0091] Carmine pigment: brand C805213, purchased from Maclean Company.

[0092] Beet red pigment: brand R854445, purchased from Maclean Company.

[0093] Methanol: analytical grade, purchased from Damas.

[0094] Ammonium acetate: analytical grade, purchased from Sinopharm.

[0095] Preparation Example 1

[0096] At room temperature, 50 μg of FSDNA and 4 μmol of AgNO3 were dissolved in 100 μL of ultrapure water to obtain a mixed solution. 10 μL of the mixed solution was dropped onto a Cu sheet for an in situ reduction reaction. After reacting for 5 minutes, the Cu sheet was washed with distilled water until no reaction liquid was left on the Cu sheet. The Cu sheet was then dried at 40°C for 20 seconds to obtain a coral-like SERS active substrate G1.

[0097] Preparation Example 2

[0098] This preparation example was carried out using the same process as Preparation Example 1, except that:

[0099] The amount of FSDNA used was 75 μg, and the other conditions were the same as those in Preparation Example 1, to prepare a coral-like SERS active substrate G2.

[0100] Preparation Example 3

[0101] This preparation example was carried out using the same process as Preparation Example 1, except that:

[0102] The amount of FSDNA used was 12.5 μg, and the other conditions were the same as those in Preparation Example 1, to prepare a coral-like SERS active substrate G3.

[0103] Preparation Example 4

[0104] This preparation example was carried out using the same process as Preparation Example 1, except that:

[0105] The amount of AgNO3 used was 5 μmol, and the other conditions were the same as those in Preparation Example 1, to prepare a coral-like SERS active substrate G4.

[0106] Preparation Example 5

[0107] This preparation example was carried out using the same process as Preparation Example 1, except that:

[0108] The amount of AgNO3 used was 6 μmol, and the other conditions were the same as those in Preparation Example 1, to prepare a coral-like SERS active substrate G5.

[0109] Preparation Example 6

[0110] This preparation example was carried out using the same process as Preparation Example 1, except that:

[0111] The in situ reduction reaction time was 10 min, and the other conditions were the same as those in Preparation Example 1, thereby obtaining a coral-like SERS active substrate G6.

[0112] Preparation Example 7

[0113] This preparation example was carried out using the same process as Preparation Example 1, except that:

[0114] The in situ reduction reaction time was 3 min, and the other conditions were the same as those in Preparation Example 1, thereby obtaining a coral-like SERS active substrate G7.

[0115] Comparative Preparation Example 1

[0116] This comparative preparation example was carried out using the same process as Preparation Example 1, except that:

[0117] 50 μg of FSDNA was replaced with 0.1 μmol of CTAC, and the other conditions were the same as those in Preparation Example 1 to prepare a SERS active substrate DG1.

[0118] Comparative Preparation Example 2

[0119] This comparative preparation example was carried out using the same process as Preparation Example 1, except that:

[0120] 50 μg of FSDNA was replaced with 0.1 μg of PVP, and the other conditions were the same as those in Preparation Example 1 to prepare a SERS active substrate DG2.

[0121] Comparative Preparation Example 3

[0122] This comparative preparation example was carried out using the same process as Preparation Example 1, except that:

[0123] 50 μg of FSDNA was replaced with 0.15 μg of BSA, and the other conditions were the same as those in Preparation Example 1 to prepare a SERS active substrate DG3.

[0124] In order to demonstrate the uniformity of the coral-like SERS active substrate provided by the present invention, ten different batches of coral-like SERS active substrates were prepared in the present invention. 30 mmol / L CA was used as the signal molecule to carry out contact reaction with the ten different batches of coral-like SERS active substrates. After reacting for 10 minutes, the substrates were dried at 40°C for 20 seconds, and three points were randomly selected for SERS detection. 20 points were randomly selected on one coral-like SERS active substrate for SERS detection. The specific test results are shown in FIG. Figure 6 The specific steps of the SERS detection are as follows:

[0125] The dried coral-like SERS active substrate was detected using a portable Raman spectrometer (SEED3000, manufactured by Shanghai Ruhai Optoelectronics Technology Co., Ltd.). The specific detection parameters were: 638 nm laser, power of about 0.5 mW, spectral scanning range of 200 cm -1 -2000cm -1 , the integration time is 1s, and the cumulative measurement is 3 times.

[0126] Figure 6 This is a data graph provided by the present invention to prove the uniformity of the SERS active substrate. Figure 5 It can be seen that Figure 6 The RSD value of the SERS intensity of ten different batches of coral-like SERS active substrates in a is 9.92%. Figure 6 The RSD value of the SERS intensity of the first batch of coral-like SERS active substrates in b is 9.39%, indicating that the coral-like SERS active substrates provided by the present invention have good uniformity and reproducibility.

[0127] In order to demonstrate the sensitivity of the coral-like SERS active substrate provided by the present invention, the present invention uses 0.1 mol / L 4-mercaptophenylboronic acid aqueous solution (4-MBPA) and 0.01 μmol / L 4-mercaptophenylboronic acid aqueous solution as probe molecules to measure the enhancement factor of the SERS active substrate. The specific test results are shown in Figure 7. The specific measurement steps are the same as above:

[0128] EF=(I SERS / C SERS )×(C Raman / I Raman )

[0129] Among them, I SERS The 0.1 mol / L 4-MBPA aqueous solution was in contact with the coral-like SERS active substrate G1 at 1070 cm -1 SERS spectrum intensity at ;

[0130] I Raman It means that 0.1 mol / L 4-MBPA aqueous solution is -1 Normal Raman spectrum intensity at ;

[0131] C SERS represents the concentration of the sampled 4-MBPA aqueous solution in the SERS measurement;

[0132] C Raman Indicates the concentration of the 4-MBPA aqueous solution sampled in ordinary Raman measurement.

[0133] Figure 7It is a Raman spectrum diagram of the SERS active substrate enhancement factor calculated according to the present invention. As can be seen from Figure 7, the enhancement factor of the SERS active substrate provided by the present invention is 1.96×10 7 , indicating that the prepared SERS active substrate has high sensitivity.

[0134] Test Example 1

[0135] At room temperature, 1 μg of allura red pigment and 0.10 μmol of CA were dissolved in 10 μL of ultrapure water. The mixture was mixed at 4000 rpm for 20 seconds and then allowed to stand for 5 minutes. The pH of the system was adjusted to 5.22 to obtain a mixture I. The mixture I was dropped onto the coral-like SERS active substrate G1 for contact reaction. After reacting for 10 minutes, the mixture was dried at 40°C for 20 seconds and subjected to SERS detection. The specific test results are shown in FIG. Figure 5 .

[0136] Test Example 2

[0137] This test case uses the same process as Test Case 1, except that:

[0138] The dosage of CA was 0.20 μmol, and the other conditions were the same as those in Test Example 1. The test results are shown in Figure 5 .

[0139] Test Example 3

[0140] This test case uses the same process as Test Case 1, except that:

[0141] The dosage of CA was 0.05 μmol, and the other conditions were the same as those in Test Example 1. The test results are shown in Figure 5 .

[0142] Test Example 4

[0143] This test case uses the same process as Test Case 1, except that:

[0144] The contact reaction time is 5 minutes, and the other conditions are the same as those in Test Example 1. The test results are shown in Figure 5 .

[0145] Test Example 5

[0146] This test case uses the same process as Test Case 1, except that:

[0147] The contact reaction time was 20 min, and the other conditions were the same as those in Test Example 1. The test results are shown in FIG5 .

[0148] Test Example 6

[0149] This test case uses the same process as Test Case 1, except that:

[0150] The rest time is 1 min, and the other conditions are the same as those in test example 1. The test results are shown in Figure 5 .

[0151] Test Example 7

[0152] This test case uses the same process as Test Case 1, except that:

[0153] The rest time is 10 minutes, and the other conditions are the same as those in test example 1. The test results are shown in Figure 5 .

[0154] Test Example 8

[0155] This test case uses the same process as Test Case 1, except that:

[0156] The pH value of the system is 2.31, and the other conditions are the same as those in Test Example 1. The test results are shown in Figure 5 .

[0157] Test Example 9

[0158] This test case uses the same process as Test Case 1, except that:

[0159] The pH value of the system is 6.20, and the other conditions are the same as those in Test Example 1. The test results are shown in Figure 5 .

[0160] Test Example 10

[0161] This test case uses the same process as Test Case 1, except that:

[0162] The weight of allura red pigment was replaced by brilliant blue pigment, and the other conditions were the same as those in test example 1. The test results are shown in Figure 8 .

[0163] Test Example 11

[0164] This test case uses the same process as Test Case 1, except that:

[0165] The weight of allura red pigment was replaced by sunset yellow pigment. The other conditions were the same as those in test example 1. The test results are shown in Figure 8 .

[0166] Comparative test example 1

[0167] This comparative test example uses the same process as Test Example 1, except that:

[0168] 1 μg of allura red pigment was replaced with 10 μg of erythrosine pigment. The other conditions were the same as those in Test Example 1. The test results are shown in Figure 8 .

[0169] Comparative test example 2

[0170] This comparative test example uses the same process as Test Example 1, except that:

[0171] 1 μg of allura red pigment was replaced with 10 μg of new red pigment. The other conditions were the same as those in Test Example 1. The test results are shown in Figure 8 .

[0172] Comparative test example 3

[0173] This comparative test example uses the same process as Test Example 1, except that:

[0174] 1 μg of allura red pigment was replaced with 10 μg of amaranth pigment. The other conditions were the same as those in Test Example 1. The test results are shown in Figure 8 .

[0175] Comparative test example 4

[0176] This comparative test example uses the same process as Test Example 1, except that:

[0177] 1 μg of allura red pigment was replaced with 10 μg of indigo pigment. The other conditions were the same as those in Test Example 1. The test results are shown in Figure 8 .

[0178] Comparative test example 5

[0179] This comparative test example uses the same process as Test Example 1, except that:

[0180] 1 μg of allura red pigment was replaced with 10 μg of anthocyanin pigment. The other conditions were the same as those in Test Example 1. The test results are shown in Figure 8 .

[0181] Comparative Test Example 6

[0182] This comparative test example uses the same process as Test Example 1, except that:

[0183] 1 μg of allura red pigment was replaced with 10 μg of curcumin pigment. The other conditions were the same as those in Test Example 1. The test results are shown in Figure 8 .

[0184] Comparative Test Example 7

[0185] This comparative test example uses the same process as Test Example 1, except that:

[0186] 1 μg of allura red pigment was replaced with 10 μg of carmine pigment. The other conditions were the same as those in Test Example 1. The test results are shown in Figure 8 .

[0187] Comparative test example 8

[0188] This comparative test example uses the same process as Test Example 1, except that:

[0189] 1 μg of allura red pigment was replaced with 10 μg of betalain pigment. The other conditions were the same as those in Test Example 1. The test results are shown in Figure 8 .

[0190] Figure 8 The Raman spectra of different food pigments detected by the method provided by the present invention are shown in FIG. Figure 8 It can be seen that the SERS signal displayed by the method provided by the present invention when detecting pigments containing sulfonic acid groups is significantly stronger than that when detecting pigments without sulfonic acid groups, indicating that the method provided by the present invention for detecting food pigments has good selectivity.

[0191] Example 1: Quantitative analysis of allura red pigment

[0192] a: Drawing of standard curve

[0193] At room temperature, 0.001 μg, 0.003 μg, 0.005 μg, 0.007 μg, 0.009 μg, 0.02 μg, 0.04 μg, 0.06 μg, 0.08 μg, and 0.1 μg of allura red pigment and 0.10 μmol of CA were dissolved in 10 μL of ultrapure water, mixed at 4000 rpm for 20 s using a vortex mixer, and then allowed to stand for 5 min. The pH of the system was adjusted to 5.22 to obtain a mixture I. The mixture I was dropped onto the coral-like SERS active substrate G1 for contact reaction. After 10 min of reaction, SERS detection was performed. The specific test results are shown in FIG. Figure 9 , and linear fitting was performed using origin software, and the obtained linear equation is shown in Table 1 below.

[0194] Table 1

[0195]

[0196]

[0197] b: Detection of samples containing allura red pigment

[0198] Beverage sample 1 (strawberry cocktail) and candy sample 2 (strawberry candy) containing allura red pigment were tested. Candy sample 2 required pretreatment, and the specific steps were as follows:

[0199] Take one pellet and place it in a 50 mL centrifuge tube, add 5 mL of distilled water, and after ultrasonic treatment for 20 min, take 1 mL of the liquid and place it in a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 10 min, and take the supernatant for later use.

[0200] 0.10 μmol of CA, 1 μL of beverage sample 1 (candy sample 2), and 8 μL of ultrapure water were mixed at 4000 rpm using a vortex mixer for 20 seconds and then allowed to stand for 5 minutes. The pH of the system was adjusted to 5.22. The mixture was then dropped onto the coral-like SERS active substrate G1 for contact reaction. After reacting for 10 minutes, the mixture was blotted dry with filter paper and subjected to SERS detection. The content of allura red pigment in beverage sample 1 (candy sample 2) was calculated according to the linear equation. The specific results are shown in Tables 4 and Figure 10 .

[0201] In order to demonstrate the accuracy of the method provided by the present invention for detecting allura red pigment in actual samples, the present application adopts high performance liquid chromatography (HPLC method) to detect the content of allura red pigment in actual samples. The specific results are shown in Table 4.

[0202] The specific detection conditions of the HPLC method are as follows: C18 column, injection volume 10 μL, flow rate 1 mL / min, column temperature 35° C., detection wavelength 504 nm, isocratic elution: methanol: ammonium acetate (0.02 mol / L) = 40:60 (v:v).

[0203] c: Recovery of allura red pigment spiked into artificial urine

[0204] The standard addition method was used to detect the content of allura red pigment added to artificial urine to evaluate the reliability of the coral-like SERS active substrate provided by the present invention for quantitative detection of pigments in biological samples. The specific steps are as follows:

[0205] Different concentrations of allura red pigment were prepared: 0.25 μg / mL, 0.55 μg / mL, 0.75 μg / mL, 3.50 μg / mL, 5.50 μg / mL, and 7.50 μg / mL of allura red pigment and 10 mmol / L of CA were added to artificial urine to obtain artificial urine spiked samples. 10 μL of the artificial urine spiked sample was dropped onto the coral-like SERS active substrate for contact reaction. After 10 minutes of reaction, SERS detection was performed. The specific results are shown in Tables 5 and Figure 11 .

[0206] Example 2: Quantitative Analysis of Sunset Yellow

[0207] a: Drawing of standard curve

[0208] At room temperature, 0.001 μg, 0.003 μg, 0.005 μg, 0.007 μg, 0.009 μg, 0.02 μg, 0.04 μg, 0.06 μg, 0.08 μg, and 0.1 μg of sunset yellow pigment and 0.10 μmol of CA were dissolved in 10 μL of ultrapure water, mixed at 4000 rpm using a vortex mixer for 20 seconds, and then allowed to stand for 5 minutes. The pH of the system was adjusted to 5.22 to obtain a mixture I. The mixture I was dropped onto the coral-like SERS active substrate G1 for contact reaction. After 10 minutes of reaction, SERS detection was performed. The specific test results are shown in FIG. Figure 9 , and linear fitting was performed using origin software, and the obtained linear equation is shown in Table 2 below.

[0209] Table 2

[0210] Linear range Linear equations <![CDATA[R 2 ]]> 0.1μg / mL-1μg / mL y=3321x+93.51 0.9969 1μg / mL-10μg / mL y=1062x+2126 0.9989

[0211] b: Detection of samples containing sunset yellow pigment

[0212] Beverage sample 3 (orange soda) and candy sample 4 (orange candy) containing sunset yellow pigment were tested. The specific testing steps were the same as in Example 1, and the content of sunset yellow pigment in beverage sample 3 (candy sample 4) was calculated according to the linear equation in Table 2. The specific results are shown in Tables 4 and Figure 10 .

[0213] In order to demonstrate the accuracy of the method provided by the present invention for detecting allura red pigment in actual samples, the present application uses high performance liquid chromatography to detect the content of allura red pigment in actual samples. The specific results are shown in Table 4.

[0214] The specific detection conditions of the HPLC method are as follows: C18 column, injection volume 10 μL, flow rate 1 mL / min, column temperature 35° C., detection wavelength 481 nm, isocratic elution: methanol: ammonium acetate (0.02 mol / L) = 40:60 (v:v).

[0215] c: Sunset yellow pigment artificial urine spike recovery

[0216] The standard addition method was used to detect the content of sunset yellow pigment added to artificial urine to evaluate the reliability of the coral-like SERS active substrate provided by the present invention for quantitative detection of pigments in biological samples. The specific steps were the same as those in Example 1. The specific results are shown in Tables 5 and Figure 11 .

[0217] Example 3: Quantitative analysis of brilliant blue pigment

[0218] a: Drawing of standard curve

[0219] At room temperature, 0.001 μg, 0.003 μg, 0.005 μg, 0.007 μg, 0.009 μg, 0.02 μg, 0.04 μg, 0.06 μg, 0.08 μg, and 0.1 μg of brilliant blue pigment and 0.10 μmol of CA were dissolved in 10 μL of ultrapure water, mixed at 4000 rpm for 20 s using a vortex mixer, and then allowed to stand for 5 min. The pH of the system was adjusted to 5.22 to obtain a mixture I. The mixture I was dropped onto the coral-like SERS active substrate G1 for contact reaction. After 10 min of reaction, SERS detection was performed. The specific test results are shown in FIG. Figure 9 , and linear fitting was performed using origin software, and the obtained linear equation is shown in Table 3 below.

[0220] Table 3

[0221] Linear range Linear equations <![CDATA[R 2 ]]> 0.1μg / mL-1μg / mL y=3958x-186.2 0.9934 1μg / mL-10μg / mL y=953.8x+2924 0.9984

[0222] b: Detection of samples containing brilliant blue pigment

[0223] Beverage sample 5 (blueberry cocktail) and beverage sample 6 (peach soda) containing brilliant blue pigment were tested. The specific testing steps were the same as those in Example 1, and the content of brilliant blue pigment in beverage sample 5 (beverage sample 6) was calculated according to the linear equation in Table 3. The specific results are shown in Tables 4 and Figure 10 .

[0224] In order to demonstrate the accuracy of the method provided by the present invention for detecting allura red pigment in actual samples, the present application uses high performance liquid chromatography to detect the content of allura red pigment in actual samples. The specific results are shown in Table 4.

[0225] The specific detection conditions of the high performance liquid chromatography method are as follows: C18 column, injection volume 10 μL, flow rate 1 mL / min, column temperature 35°C, detection wavelength 600 nm, linear gradient elution: methanol: 0-5 min, from 20% to 40%; 5-8 min, maintain at 40%; 8-10 min, from 40% to 20%; ammonium acetate (0.02 mol / L): 0-5 min, from 80% to 60%; 5-8 min, maintain at 60%; 8-10 min, from 60% to 80%.

[0226] c: Brilliant blue pigment artificial urine spiked recovery

[0227] The content of brilliant blue pigment added to artificial urine was detected by the standard addition method to evaluate the reliability of the coral-like SERS active substrate provided by the present invention in the quantitative detection of pigments in biological samples. The specific steps were the same as those in Example 1. The specific results are shown in Tables 5 and Figure 11 .

[0228] Table 4

[0229] sample SERS detection results (μg / mL) HPLC test results (μg / mL) RSD value Beverage Sample 1 0.99 1.10 0.06 Candy Sample 2 3.62 3.36 0.01 Beverage Sample 3 6.83 6.93 0.04 Sample candy 4 2.55 2.75 0.01 Beverage Sample 5 1.22 1.24 0.01 Beverage Sample 6 1.22 1.19 0.05

[0230] Table 5

[0231]

[0232] Figure 9 The Raman spectra and standard linear curves of the method provided by the present invention are used to detect different contents of food pigments. Figure 9 It can be seen that the three pigments showed good linear relationships between 0.1-1 μg / mL and 1-10 μg / mL, respectively.

[0233] Figure 10 The Raman spectra of food coloring in different actual samples are detected by the method provided by the present invention. Figure 10 It can be seen from the results that the contents of allura red, sunset yellow and brilliant blue in the actual samples detected by the SERS method provided by the present invention and the HPLC method are very similar, which proves that the method provided by the present invention can accurately detect the contents of allura red, sunset yellow and brilliant blue.

[0234] Figure 11 This is the Raman spectrum of the food coloring in artificial urine provided by the present invention. Figure 11 The results show that the SERS method provided by the present invention has good accuracy and precision in the detection of actual samples and can be applied to the quantitative detection of pigments in biological samples.

[0235] Figure 12 The length and diameter distribution diagram of silver nanoparticles on the SERS active substrate prepared with different protective agents provided by the present invention. Figure 12 It can be seen that compared with BSA, CTAC, and PVP, the diameter and length distribution range of the silver nanoparticles on the coral-like SERS active substrate prepared using FSDNA as a protective agent is better, making the prepared substrate more uniform.

[0236] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A method for preparing a coral-like SERS active substrate, characterized in that: The method includes: In the presence of a protective agent and solvent I, silver nitrate and a copper sheet are subjected to an in-situ reduction reaction to obtain the coral-like SERS active substrate; The protective agent is fish sperm DNA; the solvent I is water; Relative to 100 μL of the solvent I, the amount of the protective agent used is 50-75 μg, and the amount of the silver nitrate used is 4-5 μmol.

2. The method according to claim 1, wherein The in-situ reduction reaction conditions at least meet the following requirements: temperature of 20-30° C. and time of 3-20 min.

3. A coral-like SERS active substrate prepared by the method according to claim 1 or 2.

4. Use of the coral-shaped SERS active substrate according to claim 3 in detecting food pigments.

5. A method for determining the content of food pigments, characterized in that: The method includes: (1) in the presence of solvent II, reacting cysteamine hydrochloride with food coloring to obtain a mixture I; (2) contacting the mixture I with a coral-like SERS active substrate; (3) establishing a standard curve using SERS spectroscopy to determine the content of the food pigment; Wherein, the food coloring is selected from at least one of allura red, brilliant blue, and sunset yellow; The coral-like SERS active substrate is the coral-like SERS active substrate according to claim 3.

6. The method according to claim 5, wherein: Relative to 10 μL of the solvent II, the amount of the cysteamine hydrochloride used is 0.05-0.20 μmol, and the amount of the food coloring used is 0.001-0.1 μg.

7. The method according to claim 5 or 6, wherein: The conditions of the first reaction at least meet the following requirements: the reaction is carried out under stirring conditions, with a stirring speed of 4000-5000 rpm, a stirring time of 0.5-10 min, and a pH value of 2.30-6.

20.

8. The method according to claim 5 or 6, wherein: The contact reaction conditions at least meet the following requirements: temperature of 20-30° C. and time of 1-30 min.

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